Polypeptides having endoglucanase activity and polynucleotides encoding same

ABSTRACT

The present invention relates to isolated polypeptides having endoglucanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/090,400 filed on Apr. 16, 2008, which is a 35 U.S.C. 371 nationalapplication of PCT/EP2006/068509 filed Nov. 15, 2006, which claimspriority or the benefit under 35 U.S.C. 119 of Danish application no. PA2005 01599 filed Nov. 16, 2005 and U.S. provisional application No.60/738,430 filed Nov. 21, 2005, the contents of which are fullyincorporated herein by reference.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to isolated polypeptides havingendoglucanase activity and isolated polynucleotides encoding thepolypeptides. The invention also relates to nucleic acid constructs,vectors, and host cells comprising the polynucleotides as well asmethods for producing and using the polypeptides in the detergent, paperand pulp, oil drilling, oil extraction, wine and juice, foodingredients, animal feed or textile industries.

BACKGROUND OF THE INVENTION

Cellulose is a polymer of glucose linked by beta-1,4-glucosidic bonds.Cellulose chains form numerous intra- and intermolecular hydrogen bonds,which result in the formation of insoluble cellulose micro-fibrils.Microbial hydrolysis of cellulose to glucose involves the followingthree major classes of cellulases: (i) endoglucanases (EC 3.2.1.4) whichcleave beta-1,4-glucosidic links randomly throughout cellulosemolecules, also called endo-beta-1,4-glucanases; (ii) cellobiohydrolases(EC 3.2.1.91) which digest cellulose from the non-reducing end,releasing cellobiose; and (iii) beta-glucosidases (EC 3.2.1.21) whichhydrolyse cellobiose and low molecular-weight cellodextrins to releaseglucose.

Beta-1,4-glucosidic bonds are also present in other naturally occurringpolymers, e.g., in the beta-glucans from plants such as barley and oats.In some cases, endoglucanases also provide hydrolysis of suchnon-cellulose polymers.

Cellulases are produced by many microorganisms and are often present inmultiple forms. Recognition of the economic significance of theenzymatic degradation of cellulose has promoted an extensive search formicrobial cellulases, which can be used industrially. As a result, theenzymatic properties and the primary structures of a large number ofcellulases have been investigated. On the basis of the results of ahydrophobic cluster analysis of the amino acid sequence of the catalyticdomain, these cellulases have been placed into different families ofglycosyl hydrolases; fungal and bacterial glycosyl hydrolases have beengrouped into 35 families (Henrissat, “A classification of glycosylhydrolases based on amino acid sequence similarities”, Biochem. J. 280:309-316 (1991); Henrissat and Bairoch, “New families in theclassification of glycosyl hydrolases based on amino acid sequencesimilarities”, Biochem. J. 293: 781-788 (1993)). Most cellulases consistof a carbohydrate binding module (CBM) and a catalytic domain (CAD)separated by a linker which may be rich in proline and hydroxy aminoacid residues. Another classification of cellulases has been establishedon the basis of the similarity of their CBMs (Gilkes et al. (1991))giving five families of glycosyl hydrolases (I-V).

Cellulases are synthesized by a large number of microorganisms whichinclude fungi, actinomycetes, myxobacteria and true bacteria but also byplants. Especially endo-beta-1,4-glucanases of a wide variety ofspecificities have been identified. Many bacterial endoglucanases havebeen described (Gilbert and Hazlewood, 1993, J. Gen. Microbiol.139:187-194; Henrissat and Bairoch, “New families in the classificationof glycosyl hydrolases based on amino acid sequence similarities”,Biochem. J. 293: 781-788 (1993)).

An important industrial use of cellulolytic enzymes is for treatment ofpaper pulp, e.g., for improving the drainage or for de-inking ofrecycled paper. Another important industrial use of cellulolytic enzymesis for treatment of cellulosic textile or fabrics, e.g., as ingredientsin detergent compositions or fabric softener compositions, forbio-polishing of new fabric (garment finishing), and for obtaining a“stone-washed” look of cellulose-containing fabric, especially denim,and several methods for such treatment have been suggested, e.g., in GB1368599, EP 0307564 and EP 0435876, WO 91/17243, WO 91/10732, WO91/17244, WO 95/24471 and WO 95/26398. JP patent application no.13049/1999 discloses a heat resistant alkaline cellulase derived fromBacillus sp. KSM-S237 (deposited as FERM-P-16067) suitable fordetergents.

There is an ever existing need for providing novel cellulase enzymes orenzyme preparations which may be used for applications where cellulase,preferably an endo-beta-1,4-glucanase, activity (endoglucanase, EC3.2.1.4) is desirable.

The object of the present invention is to provide polypeptides andpolypeptide compositions having substantial beta-1,4-glucanase activityunder slightly acid to alkaline conditions and improved performance inpaper pulp processing, textile treatment, laundry processes, extractionprocesses or in animal feed; preferably such novel well-performingendo-glucanases are producible or produced by using recombinanttechniques in high yields.

SUMMARY OF THE INVENTION

The present invention relates to isolated polypeptides havingendoglucanase activity selected from the group consisting of:

(a) a polypeptide having an amino acid sequence which has at least 72%identity with amino acids 1 to 759 of SEQ ID NO: 2;

(b) a polypeptide which is encoded by a nucleotide sequence whichhybridizes under at least low stringency conditions with (i) nucleotides100 to 2376 of SEQ ID NO: 1, or (ii) a complementary strand of (i); and

(c) a variant comprising a conservative substitution, deletion, and/orinsertion of one or more amino acids of amino acids 1 to 759 of SEQ IDNO: 2.

The present invention also relates to isolated polynucleotides encodingpolypeptides having endoglucanase activity, selected from the groupconsisting of:

(a) a polynucleotide encoding a polypeptide having an amino acidsequence which has at least 72% identity with amino acids 1 to 759 ofSEQ ID NO: 2;

(b) a polynucleotide which hybridizes under at least low stringencyconditions with (i) nucleotides 100 to 2376 of SEQ ID NO: 1, or (ii) acomplementary strand of (i).

The present invention also relates to nucleic acid constructs,recombinant expression vectors, and recombinant host cells comprisingthe polynucleotides.

The present invention also relates to methods for producing suchpolypeptides having endoglucanase activity comprising (a) cultivating arecombinant host cell comprising a nucleic acid construct comprising apolynucleotide encoding the polypeptide under conditions conducive forproduction of the polypeptide; and (b) recovering the polypeptide.

The endo-beta-1,4-glucanase of the invention has stability and activityproperties that make it exceptionally well-suited for use inapplications involving aqueous alkaline solutions that containsurfactants and/or oxidative active species such as chemical bleaches.Such application conditions are very commonly found, both withinhousehold and industrial detergents, textile finishing treatments and inthe manufacture or recycling of cellulosic pulps.

Because the endoglucanase of the invention maintains its activity to anexceptional extent under such relevant application conditions it iscontemplated that it will be more useful than other known enzymes, e.g.,when used in detergents, for paper/pulp processing or for textiletreatments. The present invention thus also relates to methods of usingthe polypeptides of the invention in a detergent or textile treatmentcomposition, a composition for treatment of paper pulp or fordegradation of biomass, e.g., for the production of ethanol. Further,the invention relates to methods for washing textile, kitchenware orhard surfaces with a detergent comprising the polypeptides, methods fortreatment of cellulosic textile or fabrics, such as softening,bio-polishing or stone-washing. Also, methods for improving the drainageor for de-inking of recycled paper are included.

The present invention further relates to nucleic acid constructscomprising a gene encoding a protein, wherein the gene is operablylinked to one or both of a first nucleotide sequence encoding a signalpeptide consisting of nucleotides 1 to 99 of SEQ ID NO: 1.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1, Alignment of the amino acid sequence of the polypeptide of theinvention (ACE160, SEQ ID NO:2) with related polypeptides of the priorart. The prior art polypeptides are disclosed as:

Name Entry number Patent number KSM-64 (SEQ ID NO: 5) ADP87708, GeneseqPJP2004173598 KSM-365 (SEQ ID NO: 6) AAR77395, GeneseqP JP07203960-1994KSM-634 (SEQ ID NO: 7) AAR07478, GeneseqP JP01281090 KSM-S237 (SEQ IDNO: 8) ADP87707, GeneseqP JP2004173598 MB1181 (SEQ ID NO: 9) ABG76403,GeneseqP WO200299091 KSM-635 (SEQ ID NO: 10) P19424, Uniprot —

FIG. 2, Phylogenetic tree showing the relationship of the endoglucanaseof the invention (ACE160, SEQ ID NO:2) with prior art polypeptidesequences were constructed upon alignment with default settings in theClustalV function of program MegAlign™ version 5.05 in DNAStar™ programpackage.

DEFINITIONS

Endoglucanase activity: The term “endoglucanase activity” is definedherein as a hydrolytic activity which catalyzes the endohydrolysis of1,4-beta-D-glucosidic linkages in cellulose, lichenin and cerealbeta-D-glucans, EC 3.2.1.4. A method for determination of endoglucanaseactivity is described below.

The polypeptides of the present invention have at least 70%, morepreferably at least 80%, even more preferably at least 90%, even morepreferably at least 95%, most preferably at least 98%, and even mostpreferably at least 100% of the endoglucanase activity of thepolypeptide consisting of the amino acid sequence shown as amino acids 1to 759 of SEQ ID NO: 2, or the catalytic core domain consisting of theamino acid 65 to 347 of SEQ ID NO: 2.

Isolated polypeptide: The term “isolated polypeptide” as used hereinrefers to a polypeptide which is at least 20% pure, preferably at least40% pure, more preferably at least 60% pure, even more preferably atleast 80% pure, most preferably at least 90% pure, and even mostpreferably at least 95% pure, as determined by SDS-PAGE.

Substantially pure polypeptide: The term “substantially purepolypeptide” denotes herein a polypeptide preparation which contains atmost 10%, preferably at most 8%, more preferably at most 6%, morepreferably at most 5%, more preferably at most 4%, at most 3%, even morepreferably at most 2%, most preferably at most 1%, and even mostpreferably at most 0.5% by weight of other polypeptide material withwhich it is natively associated. It is, therefore, preferred that thesubstantially pure polypeptide is at least 92% pure, preferably at least94% pure, more preferably at least 95% pure, more preferably at least96% pure, more preferably at least 96% pure, more preferably at least97% pure, more preferably at least 98% pure, even more preferably atleast 99%, most preferably at least 99.5% pure, and even most preferably100% pure by weight of the total polypeptide material present in thepreparation.

The polypeptides of the present invention are preferably in asubstantially pure form. In particular, it is preferred that thepolypeptides are in “essentially pure form”, i.e., that the polypeptidepreparation is essentially free of other polypeptide material with whichit is natively associated. This can be accomplished, for example, bypreparing the polypeptide by means of well-known recombinant methods orby classical purification methods.

Herein, the term “substantially pure polypeptide” is synonymous with theterms “isolated polypeptide” and “polypeptide in isolated form.”

Identity: The relatedness between two amino acid sequences is describedby the parameter “identity”.

For purposes of the present invention, the alignment of two amino acidsequences is determined by using the Needle program from the EMBOSSpackage, version 2.8.0. The Needle program implements the globalalignment algorithm described in Needleman and Wunsch, 1970, J. Mol.Biol. 48: 443-453. The substitution matrix used is BLOSUM62, gap openingpenalty is 10, and gap extension penalty is 0.5.

The degree of identity between an amino acid sequence of the presentinvention (“invention sequence”; e.g., amino acids 1 to 759 of SEQ IDNO:2 or the catalytic core domain of amino acids 65 to 347 of SEQ IDNO:2) and a different amino acid sequence (“foreign sequence”) iscalculated as the number of exact matches in an alignment of the twosequences, divided by the length of the “invention sequence” or thelength of the “foreign sequence”, whichever is the shortest. The resultis expressed in percent identity.

An exact match occurs when the “invention sequence” and the “foreignsequence” have identical amino acid residues in the same positions ofthe overlap (in the alignment example below this is represented by “|”).The length of a sequence is the number of amino acid residues in thesequence (e.g., the length of the “invention sequence” of SEQ ID NO:2 is759 amino acids).

In the alignment example below, the overlap is the amino acid sequence“HTWGER.NLG” of Sequence 1; or the amino acid sequence “HGWGEDANLA” ofSequence 2. A gap is indicated by a “.”.

Alignment Example

The length of the overlap of the “invention sequence” may be at least20% of the length of the “invention sequence”, more preferably at least30%, 40%, 50%, 60%, 70%, 80%, or at least 90% of the length of the“invention sequence”.

The length of the overlap of the “foreign sequence” may be at least 20%of the length of the “foreign sequence”, more preferably at least 30%,40%, 50%, 60%, 70%, 80%, or at least 90% of the length of the “inventionsequence”.

Polypeptide Fragment: The term “polypeptide fragment” is defined hereinas a polypeptide having one or more amino acids deleted from the aminoand/or carboxyl terminus of SEQ ID NO: 2 or a homologous sequencethereof, wherein the fragment has endoglucanase activity. Preferably,the fragment contains at least 283 amino acid residues, e.g., aminoacids 65 to 347 of SEQ ID NO: 2.

Subsequence: The term “subsequence” is defined herein as a nucleotidesequence having one or more nucleotides deleted from the 5′ and/or 3′end of SEQ ID NO: 1 or a homologous sequence thereof, wherein thesubsequence encodes a polypeptide fragment having endoglucanaseactivity. Preferably, a subsequence contains at least 849 nucleotides,e.g., nucleic acids 193 to 1041 of SEQ ID NO:1.

Allelic variant: The term “allelic variant” denotes herein any of two ormore alternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

Substantially pure polynucleotide: The term “substantially purepolynucleotide” as used herein refers to a polynucleotide preparationfree of other extraneous or unwanted nucleotides and in a form suitablefor use within genetically engineered protein production systems. Thus,a substantially pure polynucleotide contains at most 10%, preferably atmost 8%, more preferably at most 6%, more preferably at most 5%, morepreferably at most 4%, more preferably at most 3%, even more preferablyat most 2%, most preferably at most 1%, and even most preferably at most0.5% by weight of other polynucleotide material with which it isnatively associated. A substantially pure polynucleotide may, however,include naturally occurring 5′ and 3′ untranslated regions, such aspromoters and terminators. It is preferred that the substantially purepolynucleotide is at least 90% pure, preferably at least 92% pure, morepreferably at least 94% pure, more preferably at least 95% pure, morepreferably at least 96% pure, more preferably at least 97% pure, evenmore preferably at least 98% pure, most preferably at least 99%, andeven most preferably at least 99.5% pure by weight. The polynucleotidesof the present invention are preferably in a substantially pure form. Inparticular, it is preferred that the polynucleotides disclosed hereinare in “essentially pure form”, i.e., that the polynucleotidepreparation is essentially free of other polynucleotide material withwhich it is natively associated. Herein, the term “substantially purepolynucleotide” is synonymous with the terms “isolated polynucleotide”and “polynucleotide in isolated form.” The polynucleotides may be ofgenomic, cDNA, RNA, semisynthetic, synthetic origin, or any combinationsthereof.

Nucleic acid construct: The term “nucleic acid construct” as used hereinrefers to a nucleic acid molecule, either single- or double-stranded,which is isolated from a naturally occurring gene or which is modifiedto contain segments of nucleic acids in a manner that would nototherwise exist in nature. The term nucleic acid construct is synonymouswith the term “expression cassette” when the nucleic acid constructcontains the control sequences required for expression of a codingsequence of the present invention.

Control sequence: The term “control sequences” is defined herein toinclude all components, which are necessary or advantageous for theexpression of a polynucleotide encoding a polypeptide of the presentinvention. Each control sequence may be native or foreign to thenucleotide sequence encoding the polypeptide. Such control sequencesinclude, but are not limited to, a leader, polyadenylation sequence,propeptide sequence, promoter, signal peptide sequence, andtranscription terminator. At a minimum, the control sequences include apromoter, and transcriptional and translational stop signals. Thecontrol sequences may be provided with linkers for the purpose ofintroducing specific restriction sites facilitating ligation of thecontrol sequences with the coding region of the nucleotide sequenceencoding a polypeptide.

Operably linked: The term “operably linked” denotes herein aconfiguration in which a control sequence is placed at an appropriateposition relative to the coding sequence of the polynucleotide sequencesuch that the control sequence directs the expression of the codingsequence of a polypeptide.

Coding sequence: When used herein the term “coding sequence” means anucleotide sequence, which directly specifies the amino acid sequence ofits protein product. The boundaries of the coding sequence are generallydetermined by an open reading frame, which usually begins with the ATGstart codon or alternative start codons such as GTG and TTG. The codingsequence may be a DNA, cDNA, or recombinant nucleotide sequence.

Expression: The term “expression” includes any step involved in theproduction of the polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

Expression vector: The term “expression vector” is defined herein as alinear or circular DNA molecule that comprises a polynucleotide encodinga polypeptide of the invention, and which is operably linked toadditional nucleotides that provide for its expression.

Host cell: The term “host cell”, as used herein, includes any cell typewhich is susceptible to transformation, transfection, transduction, andthe like with a nucleic acid construct comprising a polynucleotide ofthe present invention.

DETAILED DESCRIPTION OF THE INVENTION Polypeptides Having EndoglucanaseActivity

In a first aspect, the present invention relates to isolatedpolypeptides having an amino acid sequence which has a degree ofidentity to amino acids 1 to 759 of SEQ ID NO:2, i.e., the maturepolypeptide of at least 72%, more preferably at least 75%, morepreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, most preferably at least 95%, and even mostpreferably at least 97%, which have endoglucanase activity (hereinafter“homologous polypeptides”). In a preferred aspect, the homologouspolypeptides have an amino acid sequence which differs by ten aminoacids, preferably by five amino acids, more preferably by four aminoacids, even more preferably by three amino acids, most preferably by twoamino acids, and even most preferably by one amino acid from amino acids1 to 759 of SEQ ID NO:2.

A polypeptide of the present invention preferably comprises the aminoacid sequence of SEQ ID NO:2 or an allelic variant thereof; or afragment thereof that has endoglucanase activity. In a preferred aspect,a polypeptide comprises the amino acid sequence of SEQ ID NO:2. Inanother preferred aspect, a polypeptide consists of the amino acidsequence of SEQ ID NO:2 or an allelic variant thereof; or a fragmentthereof that has endoglucanase activity. In another preferred aspect, apolypeptide consists of the amino acid sequence of SEQ ID NO:2.

In another preferred aspect, a polypeptide comprises a catalytic coredomain in amino acids 65 to 347 of SEQ ID NO:2, or an allelic variantthereof; or a fragment thereof that has endoglucanase activity. Thepolypeptide of the catalytic core domain has an amino acid sequencewhich has a degree of identity to amino acids 65 to 347 of SEQ ID NO:2of at least 86%, more preferably at least 88%, even more preferably atleast 90%, most preferably at least 95%, and even most preferably atleast 97%. In another preferred aspect, a polypeptide comprises acatalytic core domain in amino acids 65 to 347 of SEQ ID NO:2, or anallelic variant thereof; or a fragment thereof that has endoglucanaseactivity. In another preferred aspect, a polypeptide consists of aminoacids 65 to 347 of SEQ ID NO:2.

The annotation of the catalytic core domain is based on homology tocellulases of the Glycosyl hydrolase Family 5 (Henrissat, “Aclassification of glycosyl hydrolases based on amino acid sequencesimilarities”, Biochem. J. 280: 309-316 (1991); Henrissat and Bairoch,“New families in the classification of glycosyl hydrolases based onamino acid sequence similarities”, Biochem. J. 293: 781-788 (1993);Henrissat and Bairoch, “Updating the sequence-based classification ofglycosyl hydrolases”, Biochem. J. 316: 695-696 (1996); Davies andHenrissat, “Structures and mechanisms of glycosyl hydrolases”, Structure3: 853-859 (1995); Henrissat et al., “Cellulase families revealed byhydrophobic cluster analysis”, Gene 81:83-95 (1989); Py et al.,“Cellulase EGZ of Erwinia chrysanthemi: structural organization andimportance of His98 and Glu133 residues for catalysis”, Protein Eng. 4:325-333 (1991)). The domain annotation of the catalytic core domain isavailable through afmb.cnrs-mrs.fr/CAZY/, ebi.ac.uk/interpro/,sanger.ac.uk/Software/Pfam/, or expasy.org/prosite/.

In another aspect of the invention, the polypeptide comprises acarbohydrate binding module in amino acids 368 to 569 of SEQ ID NO:2. Inanother preferred aspect the present invention relates to polypeptidescomprising a carbohydrate binding module having a degree of identity toamino acids 368 to 569 of SEQ ID NO:2 of at least 67%, more preferablyat least 70%, more preferably at least 75%, more preferably at least80%, more preferably at least 85%, even more preferably at least 90%,most preferably at least 95%, and even most preferably at least 97%. Inanother preferred aspect, a polypeptide comprises a carbohydrate bindingmodule in amino acids 368 to 569 of SEQ ID NO:2, or an allelic variantthereof; or a fragment thereof that has carbohydrate binding activity.In another preferred aspect, a polypeptide consists of amino acids 368to 569 of SEQ ID NO:2.

The carbohydrate binding module belongs to the family 17/28. Theannotation of the CBM is based on homology with known sequences,especially the CBM of KSM-635 (Ozaki et al., “Molecular cloning andnucleotide sequence of a gene for alkaline cellulase from Bacillus sp.KSM-635”, J. Gen. Microbiol. 136:1327-1334 (1990), Uniprot No. P19424),which was annotated as a CBM based on relation to the galactose bindinglike domains described in Ito et al., “Novel thioether bond revealed bya 1.7 A crystal structure of galactose oxidase”, Nature 350: 87-90(1991); Macedo-Ribeiro et al., “Crystal structures of themembrane-binding C2 domain of human coagulation factor V”, Nature 402:434-439 (1999); Himanen et al., “Crystal structure of an Ephreceptor-ephrin complex”, Nature 414: 933-938 (2001) [PUBMED:11780069][PUB00010665]; and Marintchev et al., “Solution structure of thesingle-strand break repair protein XRCC1 N-terminal domain”, Nat.Struct. Biol. 6: 884-893 (1999)). The domain annotation of thecarbohydrate binding module is available through afmb.cnrs-mrs.fr/CAZY/,ebi.ac.uk/interpro/, sanger.ac.uk/Software/Pfam/, orexpasy.org/prosite/.

In a second aspect, the present invention relates to isolatedpolypeptides having endoglucanase activity which are encoded bypolynucleotides which hybridize under very low stringency conditions,preferably low stringency conditions, more preferably medium stringencyconditions, more preferably medium-high stringency conditions, even morepreferably high stringency conditions, and most preferably very highstringency conditions with (i) nucleotides 100 to 2376 of SEQ ID NO: 1,(ii) a subsequence of (i) or (iii) a complementary strand of (i) or (ii)(Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2dedition, Cold Spring Harbor, N.Y.). A subsequence of SEQ ID NO: 1contains at least 100 contiguous nucleotides or preferably at least 200contiguous nucleotides, more preferably 300, 400, 500, 600, 700, 800,900 contiguous nucleotides or even more preferably at least 1000contiguous nucleotides. Moreover, the subsequence may encode apolypeptide fragment which has endoglucanase activity.

The nucleotide sequence of SEQ ID NO: 1 or a subsequence thereof, aswell as the amino acid sequence of SEQ ID NO: 2 or a fragment thereof,may be used to design a nucleic acid probe to identify and clone DNAencoding polypeptides having endoglucanase activity from strains ofdifferent genera or species according to methods well known in the art.In particular, such probes can be used for hybridization with thegenomic or cDNA of the genus or species of interest, following standardSouthern blotting procedures, in order to identify and isolate thecorresponding gene therein. Such probes can be considerably shorter thanthe entire sequence, but should be at least 14, preferably at least 25,more preferably at least 35, and most preferably at least 70 nucleotidesin length. It is, however, preferred that the nucleic acid probe is atleast 100 nucleotides in length. For example, the nucleic acid probe maybe at least 200 nucleotides, preferably at least 300 nucleotides, morepreferably at least 400 nucleotides, or most preferably at least 500nucleotides in length. Even longer probes may be used, e.g., nucleicacid probes which are at least 600 nucleotides, at least preferably atleast 700 nucleotides, more preferably at least 800 nucleotides, or mostpreferably at least 900 nucleotides in length. Both DNA and RNA probescan be used. The probes are typically labeled for detecting thecorresponding gene (for example, with ³²P, ³H, ³⁵S, biotin, or avidin).Such probes are encompassed by the present invention.

A genomic DNA library prepared from such other organisms may, therefore,be screened for DNA which hybridizes with the probes described above andwhich encodes a polypeptide having endoglucanase activity. Genomic orother DNA from such other organisms may be separated by agarose orpolyacrylamide gel electrophoresis, or other separation techniques. DNAfrom the libraries or the separated DNA may be transferred to andimmobilized on nitrocellulose or other suitable carrier material. Inorder to identify a clone or DNA which is homologous with SEQ ID NO:1 ora subsequence thereof, the carrier material is used in a Southern blot.

For purposes of the present invention, hybridization indicates that thenucleotide sequence hybridizes to a labeled nucleic acid probecorresponding to the nucleotide sequence shown in SEQ ID NO:1, itscomplementary strand, or a subsequence thereof, under very low to veryhigh stringency conditions. Molecules to which the nucleic acid probehybridizes under these conditions can be detected using X-ray film.

In a preferred aspect, the nucleic acid probe is nucleotides 193 to 1041of SEQ ID NO:1. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence which encodes the polypeptide of SEQ ID NO:2, ora subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO:1. In another preferred aspect, the nucleic acidprobe is the mature polypeptide coding region of SEQ ID NO:1.

For long probes of at least 100 nucleotides in length, very low to veryhigh stringency conditions are defined as prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and either 25% formamide for very lowand low stringencies, 35% formamide for medium and medium-highstringencies, or 50% formamide for high and very high stringencies,following standard Southern blotting procedures for 12 to 24 hoursoptimally.

For long probes of at least 100 nucleotides in length, the carriermaterial is finally washed three times each for 15 minutes using 2×SSC,0.2% SDS preferably at least at 45° C. (very low stringency), morepreferably at least at 50° C. (low stringency), more preferably at leastat 55° C. (medium stringency), more preferably at least at 60° C.(medium-high stringency), even more preferably at least at 65° C. (highstringency), and most preferably at least at 70° C. (very highstringency). Preferably, the wash is conducted using 0.2×SSC, 0.2% SDSpreferably at least at 45° C. (very low stringency), more preferably atleast at 50° C. (low stringency), more preferably at least at 55° C.(medium stringency), more preferably at least at 60° C. (medium-highstringency), even more preferably at least at 65° C. (high stringency),and most preferably at least at 70° C. (very high stringency).

For short probes which are about 15 nucleotides to about 70 nucleotidesin length, stringency conditions are defined as prehybridization,hybridization, and washing post-hybridization at about 5° C. to about10° C. below the calculated T_(m) using the calculation according toBolton and McCarthy (1962, Proceedings of the National Academy ofSciences USA 48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA,0.5% NP-40, 1×Denhardt's solution, 1 mM sodium pyrophosphate, 1 mMsodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per mlfollowing standard Southern blotting procedures.

For short probes which are about 15 nucleotides to about 70 nucleotidesin length, the carrier material is washed once in 6×SCC plus 0.1% SDSfor 15 minutes and twice each for 15 minutes using 6×SSC at 5° C. to 10°C. below the calculated T_(m).

In a third aspect, the present invention relates to isolatedpolypeptides having endoglucanase activity encoded by a polynucleotidecomprising nucleotides 193 to 1041 of SEQ ID NO: 1, as a unique motif.

In a fourth aspect, the present invention relates to isolatedpolypeptides having the following physicochemical properties: pl of 4.4,pH optimum of 9, temperature optimum of 40° C. and stability at pH from5 to 10.5. The beta-1,4-glucanase of the invention is not significantlyinactivated by Fe(II) ions. A sensitivity of the enzymatic activity ofthe polypeptide to the presence of ferrous ions could place restrictionson the applicability of the polypeptide, such as in processes takingplace in metal containers or equipment.

In a fifth aspect, the present invention relates to artificial variantscomprising a conservative substitution, deletion, and/or insertion ofone or more amino acids of SEQ ID NO: 2 or the mature polypeptidethereof. Preferably, amino acid changes are of a minor nature, that isconservative amino acid substitutions or insertions that do notsignificantly affect the folding and/or activity of the protein; smalldeletions, typically of one to about 30 amino acids; small amino- orcarboxyl-terminal extensions, such as an amino-terminal methionineresidue; a small linker peptide of up to about 20-25 residues; or asmall extension that facilitates purification by changing net charge oranother function, such as a poly-histidine tract, an antigenic epitopeor a binding domain.

Examples of conservative substitutions are within the group of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions which do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. The mostcommonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser,Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg,Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.

In addition to the 20 standard amino acids, non-standard amino acids(such as 4-hydroxyproline, 6-N-methyl lysine, 2-aminoisobutyric acid,isovaline, and alpha-methyl serine) may be substituted for amino acidresidues of a wild-type polypeptide. A limited number ofnon-conservative amino acids, amino acids that are not encoded by thegenetic code, and unnatural amino acids may be substituted for aminoacid residues. “Unnatural amino acids” have been modified after proteinsynthesis, and/or have a chemical structure in their side chain(s)different from that of the standard amino acids. Unnatural amino acidscan be chemically synthesized, and preferably, are commerciallyavailable, and include pipecolic acid, thiazolidine carboxylic acid,dehydroproline, 3- and 4-methylproline, and 3,3-dimethylproline.

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide, alter the substrate specificity, change the pH optimum, andthe like.

Essential amino acids in the parent polypeptide can be identifiedaccording to procedures known in the art, such as site-directedmutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989,Science 244: 1081-1085). In the latter technique, single alaninemutations are introduced at every residue in the molecule, and theresultant mutant molecules are tested for biological activity (i.e.,endoglucanase activity) to identify amino acid residues that arecritical to the activity of the molecule. See also, Hilton et al., 1996,J. Biol. Chem. 271: 4699-4708. The active site of the enzyme or otherbiological interaction can also be determined by physical analysis ofstructure, as determined by such techniques as nuclear magneticresonance, crystallography, electron diffraction, or photoaffinitylabeling, in conjunction with mutation of putative contact site aminoacids. See, for example, de Vos et al., 1992, Science 255: 306-312;Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992,FEBS Lett. 309:59-64. The identities of essential amino acids can alsobe inferred from analysis of identities with polypeptides which arerelated to a polypeptide according to the invention.

Single or multiple amino acid substitutions can be made and tested usingknown methods of mutagenesis, recombination, and/or shuffling, followedby a relevant screening procedure, such as those disclosed byReidhaar-Olson and Sauer, 1988, Science 241: 53-57; Bowie and Sauer,1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413; or WO95/22625. Other methods that can be used include error-prone PCR, phagedisplay (e.g., Lowman et al., 1991, Biochem. 30:10832-10837; U.S. Pat.No. 5,223,409; WO 92/06204), and region-directed mutagenesis (Derbyshireet al., 1986, Gene 46:145; Ner et al., 1988, DNA 7:127).

Sources of Polypeptides Having Endoglucanase Activity

A polypeptide of the present invention may be obtained frommicroorganisms of any genus. For purposes of the present invention, theterm “obtained from” as used herein in connection with a given sourceshall mean that the polypeptide encoded by a nucleotide sequence isproduced by the source or by a strain in which the nucleotide sequencefrom the source has been inserted. In a preferred aspect, thepolypeptide obtained from a given source is secreted extracellularly.

A polypeptide of the present invention may be a bacterial polypeptide.For example, the polypeptide may be a gram positive bacterialpolypeptide such as a Bacillus polypeptide, e.g., a Bacillusalkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacilluscirculans, Bacillus coagulans, Bacillus lautus, Bacillus lentus,Bacillus licheniformis, Bacillus megaterium, Bacillusstearothermophilus, Bacillus subtilis, or Bacillus thuringiensispolypeptide; or a Streptomyces polypeptide, e.g., a Streptomyceslividans or Streptomyces murinus polypeptide; or a gram negativebacterial polypeptide, e.g., an E. coli or a Pseudomonas sp.polypeptide.

In another preferred aspect, the polypeptide is a Bacillus sp. ACE160polypeptide e.g., the polypeptide of SEQ ID NO:2.

It will be understood that for the aforementioned species, the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS),and Agricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

Furthermore, such polypeptides may be identified and obtained from othersources including microorganisms isolated from nature (e.g., soil,composts, water, etc.) using the above-mentioned probes. Techniques forisolating microorganisms from natural habitats are well known in theart. The polynucleotide may then be obtained by similarly screening agenomic or cDNA library of another microorganism. Once a polynucleotidesequence encoding a polypeptide has been detected with the probe(s), thepolynucleotide can be isolated or cloned by utilizing techniques whichare well known to those of ordinary skill in the art (see, e.g.,Sambrook et al., 1989, supra).

Polypeptides of the present invention also include fused polypeptides orcleavable fusion polypeptides in which another polypeptide is fused atthe N-terminus or the C-terminus of the polypeptide or fragment thereof.A fused polypeptide is produced by fusing a nucleotide sequence (or aportion thereof) encoding another polypeptide to a nucleotide sequence(or a portion thereof) of the present invention. Techniques forproducing fusion polypeptides are known in the art, and include ligatingthe coding sequences encoding the polypeptides so that they are in frameand that expression of the fused polypeptide is under control of thesame promoter(s) and terminator.

Polynucleotides

The present invention also relates to isolated polynucleotides having anucleotide sequence which encode a polypeptide of the present invention.In a preferred aspect, the nucleotide sequence is set forth in SEQ IDNO:1. In another preferred aspect, the nucleotide sequence is the maturepolypeptide coding region of SEQ ID NO:1. The present invention alsoencompasses nucleotide sequences which encode a polypeptide having theamino acid sequence of SEQ ID NO:2 or the mature polypeptide thereof,which differs from SEQ ID NO:1 by virtue of the degeneracy of thegenetic code. The present invention also relates to subsequences of SEQID NO:1 which encode fragments of SEQ ID NO:2 that have endoglucanaseactivity, such as the catalytic core domain of amino acid 65 to 347 ofSEQ ID NO:2 or the fragment of amino acid 368 to 569 of SEQ ID NO:2.

The present invention also relates to mutant polynucleotides comprisingat least one mutation in the mature polypeptide coding sequence of SEQID NO:1, in which the mutant nucleotide sequence encodes a polypeptidewhich consists of amino acids 1 to 759 of SEQ ID NO:2.

The techniques used to isolate or clone a polynucleotide encoding apolypeptide are known in the art and include isolation from genomic DNA,preparation from cDNA, or a combination thereof. The cloning of thepolynucleotides of the present invention from such genomic DNA can beeffected, e.g., by using the well known polymerase chain reaction (PCR)or antibody screening of expression libraries to detect cloned DNAfragments with shared structural features. See, e.g., Innis et al.,1990, PCR: A Guide to Methods and Application, Academic Press, New York.Other nucleic acid amplification procedures such as ligase chainreaction (LCR), ligated activated transcription (LAT) and nucleotidesequence-based amplification (NASBA) may be used. The polynucleotidesmay be cloned from a strain of Bacillus, or another or related organismand thus, for example, may be an allelic or species variant of thepolypeptide encoding region of the nucleotide sequence.

The present invention also relates to polynucleotides having nucleotidesequences which have a degree of identity to the mature polypeptidecoding sequence of SEQ ID NO:1 (i.e., nucleotides 100 to 2376) of atleast 60%, preferably at least 65%, more preferably at least 70%, morepreferably at least 75%, more preferably at least 80%, more preferablyat least 85%, more preferably at least 90%, even more preferably atleast 95%, and most preferably at least 97% identity, which encode anactive polypeptide.

Modification of a nucleotide sequence encoding a polypeptide of thepresent invention may be necessary for the synthesis of polypeptidessubstantially similar to the polypeptide. The term “substantiallysimilar” to the polypeptide refers to non-naturally occurring forms ofthe polypeptide. These polypeptides may differ in some engineered wayfrom the polypeptide isolated from its native source, e.g., artificialvariants that differ in specific activity, thermostability, pH optimum,or the like. The variant sequence may be constructed on the basis of thenucleotide sequence presented as the polypeptide encoding region of SEQID NO:1, e.g., a subsequence thereof, and/or by introduction ofnucleotide substitutions which do not give rise to another amino acidsequence of the polypeptide encoded by the nucleotide sequence, butwhich correspond to the codon usage of the host organism intended forproduction of the enzyme, or by introduction of nucleotide substitutionswhich may give rise to a different amino acid sequence. For a generaldescription of nucleotide substitution, see, e.g., Ford et al., 1991,Protein Expression and Purification 2: 95-107.

It will be apparent to those skilled in the art that such substitutionscan be made outside the regions critical to the function of the moleculeand still result in an active polypeptide. Amino acid residues essentialto the activity of the polypeptide encoded by an isolated polynucleotideof the invention, and therefore preferably not subject to substitution,may be identified according to procedures known in the art, such assite-directed mutagenesis or alanine-scanning mutagenesis (see, e.g.,Cunningham and Wells, 1989, Science 244: 1081-1085). In the lattertechnique, mutations are introduced at every positively charged residuein the molecule, and the resultant mutant molecules are tested forendoglucanase activity to identify amino acid residues that are criticalto the activity of the molecule. Sites of substrate-enzyme interactioncan also be determined by analysis of the three-dimensional structure asdetermined by such techniques as nuclear magnetic resonance analysis,crystallography or photoaffinity labelling (see, e.g., de Vos et al.,1992, Science 255: 306-312; Smith et al., 1992, Journal of MolecularBiology 224: 899-904; Wlodaver et al., 1992, FEBS Letters 309: 59-64).

The present invention also relates to isolated polynucleotides encodinga polypeptide of the present invention, which hybridize under very lowstringency conditions, preferably low stringency conditions, morepreferably medium stringency conditions, more preferably medium-highstringency conditions, even more preferably high stringency conditions,and most preferably very high stringency conditions with (i) nucleotides100 to 2376 of SEQ ID NO:1, (ii) nucleotides 193 to 1041 of SEQ ID NO:1,(iii) nucleotides 1104 to 1707 of SEQ ID NO:1 or (iv) a complementarystrand of (i) to (iii); or allelic variants and subsequences thereof(Sambrook et al., 1989, supra), as defined herein.

The present invention also relates to isolated polynucleotides obtainedby (a) hybridizing a population of DNA under very low, low, medium,medium-high, high, or very high stringency conditions with (i)nucleotides 100 to 2376 of SEQ ID NO:1, or (ii) a complementary strandof (i); and (b) isolating the hybridizing polynucleotide, which encodesa polypeptide having endoglucanase activity.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisingan isolated polynucleotide of the present invention operably linked toone or more control sequences which direct the expression of the codingsequence in a suitable host cell under conditions compatible with thecontrol sequences.

An isolated polynucleotide encoding a polypeptide of the presentinvention may be manipulated in a variety of ways to provide forexpression of the polypeptide. Manipulation of the polynucleotide'ssequence prior to its insertion into a vector may be desirable ornecessary depending on the expression vector. The techniques formodifying polynucleotide sequences utilizing recombinant DNA methods arewell known in the art.

The control sequence may be an appropriate promoter sequence, anucleotide sequence which is recognized by a host cell for expression ofa polynucleotide encoding a polypeptide of the present invention. Thepromoter sequence contains transcriptional control sequences whichmediate the expression of the polypeptide. The promoter may be anynucleotide sequence which shows transcriptional activity in the hostcell of choice including mutant, truncated, and hybrid promoters, andmay be obtained from genes encoding extracellular or intracellularpolypeptides either homologous or heterologous to the host cell.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention, especially in abacterial host cell, are the promoters obtained from the E. coli lacoperon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilislevansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM),Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacilluslicheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylBgenes, and prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978,Proceedings of the National Academy of Sciences USA 75: 3727-3731), aswell as the tac promoter (DeBoer et al., 1983, Proceedings of theNational Academy of Sciences USA 80: 21-25). Further promoters aredescribed in “Useful proteins from recombinant bacteria” in ScientificAmerican, 1980, 242: 74-94; and in Sambrook et al., 1989, supra.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus oryzaeTAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus nigerneutral alpha-amylase, Aspergillus niger acid stable alpha-amylase,Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucormiehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzaetriose phosphate isomerase, Aspergillus nidulans acetamidase, Fusariumvenenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Daria (WO00/56900), Fusarium venenatum Quinn (WO 00/56900), Fusarium oxysporumtrypsin-like protease (WO 96/00787), Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei endoglucanase I, Trichoderma reesei endoglucanase II, Trichodermareesei endoglucanase III, Trichoderma reesei endoglucanase IV,Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I,Trichoderma reesei xylanase II, Trichoderma reesei beta-xylosidase, aswell as the NA2-tpi promoter (a hybrid of the promoters from the genesfor Aspergillus niger neutral alpha-amylase and Aspergillus oryzaetriose phosphate isomerase); and mutant, truncated, and hybrid promotersthereof.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydro-genase (ADH1,ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionine (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a suitable transcription terminatorsequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the 3′terminus of the nucleotide sequence encoding the polypeptide. Anyterminator which is functional in the host cell of choice may be used inthe present invention.

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus oryzae TAKA amylase, Aspergillus nigerglucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillusniger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be a suitable leader sequence, anontranslated region of an mRNA which is important for translation bythe host cell. The leader sequence is operably linked to the 5′ terminusof the nucleotide sequence encoding the polypeptide. Any leader sequencethat is functional in the host cell of choice may be used in the presentinvention.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′ terminus of the nucleotide sequence and which,when transcribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencewhich is functional in the host cell of choice may be used in thepresent invention.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus oryzae TAKA amylase,Aspergillus niger glucoamylase, Aspergillus nidulans anthranilatesynthase, Fusarium oxysporum trypsin-like protease, and Aspergillusniger alpha-glucosidase.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Molecular Cellular Biology 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatcodes for an amino acid sequence linked to the amino terminus of apolypeptide and directs the encoded polypeptide into the cell'ssecretory pathway. The 5′ end of the coding sequence of the nucleotidesequence may inherently contain a signal peptide coding region naturallylinked in translation reading frame with the segment of the codingregion which encodes the secreted polypeptide. Alternatively, the 5′ endof the coding sequence may contain a signal peptide coding region whichis foreign to the coding sequence. The foreign signal peptide codingregion may be required where the coding sequence does not naturallycontain a signal peptide coding region. Alternatively, the foreignsignal peptide coding region may simply replace the natural signalpeptide coding region in order to enhance secretion of the polypeptide.However, any signal peptide coding region which directs the expressedpolypeptide into the secretory pathway of a host cell of choice may beused in the present invention.

Effective signal peptide coding regions for bacterial host cells are thesignal peptide coding regions obtained from the genes for Bacillus NCIB11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase,Bacillus licheniformis subtilisin, Bacillus licheniformisbeta-lactamase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding regions for filamentous fungal hostcells are the signal peptide coding regions obtained from the genes forAspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase,Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase,Humicola insolens cellulase, and Humicola lanuginosa lipase.

In a preferred aspect, the signal peptide coding region is nucleotides 1to 99 of SEQ ID NO:1 which encode amino acids −33 to −1 of SEQ ID NO:2.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding regions are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding region that codesfor an amino acid sequence positioned at the amino terminus of apolypeptide. The resultant polypeptide is known as a proenzyme orpropolypeptide (or a zymogen in some cases). A propolypeptide isgenerally inactive and can be converted to a mature active polypeptideby catalytic or autocatalytic cleavage of the propeptide from thepropolypeptide. The propeptide coding region may be obtained from thegenes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilisneutral protease (nprT), Saccharomyces cerevisiae alpha-factor,Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophilalaccase (WO 95/33836).

Where both signal peptide and propeptide regions are present at theamino terminus of a polypeptide, the propeptide region is positionednext to the amino terminus of a polypeptide and the signal peptideregion is positioned next to the amino terminus of the propeptideregion.

It may also be desirable to add regulatory sequences which allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those which causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. Regulatory systems in prokaryotic systems include the lac,tac, and trp operator systems. In yeast, the ADH2 system or GAL1 systemmay be used. In filamentous fungi, the TAKA alpha-amylase promoter,Aspergillus niger glucoamylase promoter, and Aspergillus oryzaeglucoamylase promoter may be used as regulatory sequences. Otherexamples of regulatory sequences are those which allow for geneamplification. In eukaryotic systems, these include the dihydrofolatereductase gene which is amplified in the presence of methotrexate, andthe metallothionein genes which are amplified with heavy metals. Inthese cases, the nucleotide sequence encoding the polypeptide would beoperably linked with the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a polynucleotide of the present invention, a promoter, andtranscriptional and translational stop signals. The various nucleicacids and control sequences described above may be joined together toproduce a recombinant expression vector which may include one or moreconvenient restriction sites to allow for insertion or substitution ofthe nucleotide sequence encoding the polypeptide at such sites.Alternatively, a nucleotide sequence of the present invention may beexpressed by inserting the nucleotide sequence or a nucleic acidconstruct comprising the sequence into an appropriate vector forexpression. In creating the expression vector, the coding sequence islocated in the vector so that the coding sequence is operably linkedwith the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) which can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the nucleotide sequence. The choice ofthe vector will typically depend on the compatibility of the vector withthe host cell into which the vector is to be introduced. The vectors maybe linear or closed circular plasmids.

The vector may be an autonomously replicating vector, i.e., a vectorwhich exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one which, when introduced into thehost cell, is integrated into the genome and replicated together withthe chromosome(s) into which it has been integrated. Furthermore, asingle vector or plasmid or two or more vectors or plasmids whichtogether contain the total DNA to be introduced into the genome of thehost cell, or a transposon may be used.

The vectors of the present invention preferably contain one or moreselectable markers which permit easy selection of transformed cells. Aselectable marker is a gene the product of which provides for biocide orviral resistance, resistance to heavy metals, prototrophy to auxotrophs,and the like.

A conditionally essential gene may function as a non-antibioticselectable marker. Non-limiting examples of bacterial conditionallyessential non-antibiotic selectable markers are the dal genes fromBacillus subtilis, Bacillus licheniformis, or other Bacilli, that areonly essential when the bacterium is cultivated in the absence ofD-alanine. Also the genes encoding enzymes involved in the turnover ofUDP-galactose can function as conditionally essential markers in a cellwhen the cell is grown in the presence of galactose or grown in a mediumwhich gives rise to the presence of galactose. Non-limiting examples ofsuch genes are those from B. subtilis or B. licheniformis encodingUTP-dependent phosphorylase (EC 2.7.7.10), UDP-glucose-dependenturidylyltransferase (EC 2.7.7.12), or UDP-galactose epimerase (EC5.1.3.2). Also a xylose isomerase gene such as xylA, of Bacilli can beused as selectable markers in cells grown in minimal medium with xyloseas sole carbon source. The genes necessary for utilizing gluconate,gntK, and gntP can also be used as selectable markers in cells grown inminimal medium with gluconate as sole carbon source. Other examples ofconditionally essential genes are known in the art. Antibioticselectable markers confer antibiotic resistance to such antibiotics asampicillin, kanamycin, chloramphenicol, erythromycin, tetracycline,neomycin, hygromycin or methotrexate.

Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3,TRP1, and URA3. Selectable markers for use in a filamentous fungal hostcell include, but are not limited to, amdS (acetamidase), argB(ornithine carbamoyltransferase), bar (phosphinothricinacetyltransferase), hph (hygromycin phosphotransferase), niaD (nitratereductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfateadenyltransferase), and trpC (anthranilate synthase), as well asequivalents thereof. Preferred for use in an Aspergillus cell are theamdS and pyrG genes of Aspergillus nidulans or Aspergillus oryzae andthe bar gene of Streptomyces hygroscopicus.

The vectors of the present invention preferably contain an element(s)that permits integration of the vector into the host cell's genome orautonomous replication of the vector in the cell independent of thegenome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the polypeptide or any other elementof the vector for integration into the genome by homologous ornonhomologous recombination. Alternatively, the vector may containadditional nucleotide sequences for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should preferably contain asufficient number of nucleic acids, such as 100 to 10,000 base pairs,preferably 400 to 10,000 base pairs, and most preferably 800 to 10,000base pairs, which have a high degree of identity with the correspondingtarget sequence to enhance the probability of homologous recombination.The integrational elements may be any sequence that is homologous withthe target sequence in the genome of the host cell. Furthermore, theintegrational elements may be non-encoding or encoding nucleotidesequences. On the other hand, the vector may be integrated into thegenome of the host cell by non-homologous recombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication which functions in a cell.The term “origin of replication” or “plasmid replicator” is definedherein as a nucleotide sequence that enables a plasmid or vector toreplicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAMβ1 permittingreplication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANS1 (Gems et al., 1991, Gene 98:61-67; Cullen et al.,1987, Nucleic Acids Research 15: 9163-9175; WO 00/24883). Isolation ofthe AMA1 gene and construction of plasmids or vectors comprising thegene can be accomplished according to the methods disclosed in WO00/24883.

More than one copy of a polynucleotide of the present invention may beinserted into the host cell to increase production of the gene product.An increase in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisinga polynucleotide of the present invention, which are advantageously usedin the recombinant production of the polypeptides. A vector comprising apolynucleotide of the present invention is introduced into a host cellso that the vector is maintained as a chromosomal integrant or as aself-replicating extra-chromosomal vector as described earlier. The term“host cell” encompasses any progeny of a parent cell that is notidentical to the parent cell due to mutations that occur duringreplication. The choice of a host cell will to a large extent dependupon the gene encoding the polypeptide and its source.

The host cell may be a unicellular microorganism, e.g., a prokaryote, ora non-unicellular microorganism, e.g., a eukaryote.

Useful unicellular microorganisms are bacterial cells such as grampositive bacteria including, but not limited to, a Bacillus cell, e.g.,Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis,Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacilluslautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium,Bacillus stearothermophilus, Bacillus subtilis, and Bacillusthuringiensis; or a Streptomyces cell, e.g., Streptomyces lividans andStreptomyces murinus, or gram negative bacteria such as E. coli andPseudomonas sp. In a preferred aspect, the bacterial host cell is aBacillus lentus, Bacillus licheniformis, Bacillus stearothermophilus, orBacillus subtilis cell. In another preferred aspect, the Bacillus cellis an alkalophilic Bacillus.

The introduction of a vector into a bacterial host cell may, forinstance, be effected by protoplast transformation (see, e.g., Chang andCohen, 1979, Molecular General Genetics 168: 111-115), using competentcells (see, e.g., Young and Spizizin, 1961, Journal of Bacteriology 81:823-829, or Dubnau and Davidoff-Abelson, 1971, Journal of MolecularBiology 56: 209-221), electroporation (see, e.g., Shigekawa and Dower,1988, Biotechniques 6: 742-751), or conjugation (see, e.g., Koehler andThorne, 1987, Journal of Bacteriology 169: 5771-5278).

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

In a preferred aspect, the host cell is a fungal cell. “Fungi” as usedherein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota,and Zygomycota (as defined by Hawksworth et al., In, Ainsworth andBisby's Dictionary of The Fungi, 8th edition, 1995, CAB International,University Press, Cambridge, UK) as well as the Oomycota (as cited inHawksworth et al., 1995, supra, page 171) and all mitosporic fungi(Hawksworth et al., 1995, supra).

In a more preferred aspect, the fungal host cell is a yeast cell.“Yeast” as used herein includes ascosporogenous yeast (Endomycetales),basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti(Blastomycetes). Since the classification of yeast may change in thefuture, for the purposes of this invention, yeast shall be defined asdescribed in Biology and Activities of Yeast (Skinner, F. A., Passmore,S. M., and Davenport, R. R., eds, Soc. App. Bacteriol. Symposium SeriesNo. 9, 1980).

In an even more preferred aspect, the yeast host cell is a Candida,Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, orYarrowia cell.

In a most preferred aspect, the yeast host cell is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensisor Saccharomyces oviformis cell. In another most preferred aspect, theyeast host cell is a Kluyveromyces lactis cell. In another mostpreferred aspect, the yeast host cell is a Yarrowia lipolytica cell.

In another more preferred aspect, the fungal host cell is a filamentousfungal cell. “Filamentous fungi” include all filamentous forms of thesubdivision Eumycota and Oomycota (as defined by Hawksworth et al.,1995, supra). The filamentous fungi are generally characterized by amycelial wall composed of chitin, cellulose, glucan, chitosan, mannan,and other complex polysaccharides. Vegetative growth is by hyphalelongation and carbon catabolism is obligately aerobic. In contrast,vegetative growth by yeasts such as Saccharomyces cerevisiae is bybudding of a unicellular thallus and carbon catabolism may befermentative.

In an even more preferred aspect, the filamentous fungal host cell is anAcremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis,Coprinus, Coriolus, Cryptococcus, Filobasidium, Fusarium, Humicola,Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora,Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

In a most preferred aspect, the filamentous fungal host cell is anAspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus,Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger orAspergillus oryzae cell. In another most preferred aspect, thefilamentous fungal host cell is a Fusarium bactridioides, Fusariumcerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, or Fusariumvenenatum cell. In another most preferred aspect, the filamentous fungalhost cell is a Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsisaneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens,Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa,or Ceriporiopsis subvermispora, Coprinus cinereus, Coriolus hirsutus,Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthorathermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaetechrysosporium, Phlebia radiata, Pleurotus eryngii, Thielavia terrestris,Trametes villosa, Trametes versicolor, Trichoderma harzianum,Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei,or Trichoderma viride strain cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238 023 and Yelton et al., 1984, Proceedings of the NationalAcademy of Sciences USA 81: 1470-1474. Suitable methods for transformingFusarium species are described by Malardier et al., 1989, Gene 78:147-156, and WO 96/00787. Yeast may be transformed using the proceduresdescribed by Becker and Guarente, In Abelson, J. N. and Simon, M. I.,editors, Guide to Yeast Genetics and Molecular Biology, Methods inEnzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Itoet al., 1983, Journal of Bacteriology 153: 163; and Hinnen et al., 1978,Proceedings of the National Academy of Sciences USA 75: 1920.

Methods of Production

The present invention also relates to methods for producing apolypeptide of the present invention, comprising (a) cultivating a cell,which in its wild-type form is capable of producing the polypeptide,under conditions conducive for production of the polypeptide; and (b)recovering the polypeptide. Preferably, the cell is of the genusBacillus.

The present invention also relates to methods for producing apolypeptide of the present invention, comprising (a) cultivating a hostcell under conditions conducive for production of the polypeptide; and(b) recovering the polypeptide.

The present invention also relates to methods for producing apolypeptide of the present invention, comprising (a) cultivating a hostcell under conditions conducive for production of the polypeptide,wherein the host cell comprises a mutant nucleotide sequence having atleast one mutation in the mature polypeptide coding region of SEQ ID NO:1, wherein the mutant nucleotide sequence encodes a polypeptide whichcomprises amino acids 1-759 of SEQ ID NO:2, or amino acids 65 to 347 ofSEQ ID NO:2 or amino acids 368 to 569 of SEQ ID NO:2, and (b) recoveringthe polypeptide.

In the production methods of the present invention, the cells arecultivated in a nutrient medium suitable for production of thepolypeptide using methods well known in the art. For example, the cellmay be cultivated by shake flask cultivation, and small-scale orlarge-scale fermentation (including continuous, batch, fed-batch, orsolid state fermentations) in laboratory or industrial fermentorsperformed in a suitable medium and under conditions allowing thepolypeptide to be expressed and/or isolated. The cultivation takes placein a suitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted, it can be recovered from cell lysates.

The polypeptides may be detected using methods known in the art that arespecific for the polypeptides. These detection methods may include useof specific antibodies, formation of an enzyme product, or disappearanceof an enzyme substrate. For example, an enzyme assay may be used todetermine the activity of the polypeptide as described herein.

The resulting polypeptide may be recovered using methods known in theart. For example, the polypeptide may be recovered from the nutrientmedium by conventional procedures including, but not limited to,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

The polypeptides of the present invention may be purified by a varietyof procedures known in the art including, but not limited to,chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing), differential solubility (e.g.,ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g.,Protein Purification, J.-C. Janson and Lars Ryden, editors, VCHPublishers, New York, 1989).

The present invention also relates to isolated enzymes havingendo-beta-1,4-glucanse activity and which are produced by one of theabove mentioned methods, preferably by recombinant productiontechniques. The isolated enzymes are preferably free from homologousimpurities. Such impurities may arise from endogenousendo-beta-1,4-glucanse genes, hence if production is performed in a hostcell which does not express endogenous polypeptides withendo-beta-1,4-glucanse activity, the enzyme will be free of homologousimpurities.

Compositions

The present invention also relates to compositions comprising apolypeptide of the present invention. Preferably, the compositions areenriched in such a polypeptide. The term “enriched” indicates that theendoglucanase activity of the composition has been increased, e.g., withan enrichment factor of 1.1.

The composition may comprise a polypeptide of the present invention asthe major enzymatic component, e.g., a mono-component composition.Alternatively, the composition may comprise multiple enzymaticactivities, such as an aminopeptidase, amylase, carbohydrase,carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextringlycosyltransferase, deoxyribonuclease, esterase, alpha-galactosidase,beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase,haloperoxidase, invertase, laccase, lipase, mannosidase, oxidase,pectinolytic enzyme, peptidoglutaminase, peroxidase, phytase,polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase,or xylanase. The additional enzyme(s) may be produced, for example, by amicroorganism belonging to the genus Aspergillus, preferably Aspergillusaculeatus, Aspergillus awamori, Aspergillus fumigatus, Aspergillusfoetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillusniger, or Aspergillus oryzae; Fusarium, preferably Fusariumbactridioides, Fusarium cerealis, Fusarium crookwellense, Fusariumculmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium sulphureum, Fusarium toruloseum, Fusarium trichothecioides, orFusarium venenatum; Humicola, preferably Humicola insolens or Humicolalanuginosa; or Trichoderma, preferably Trichoderma harzianum,Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei,or Trichoderma viride.

The polypeptide compositions may be prepared in accordance with methodsknown in the art and may be in the form of a liquid or a drycomposition. For instance, the polypeptide composition may be in theform of a granulate or a microgranulate. The polypeptide to be includedin the composition may be stabilized in accordance with methods known inthe art.

Examples are given below of preferred uses of the polypeptidecompositions of the invention. The dosage of the polypeptide compositionof the invention and other conditions under which the composition isused may be determined on the basis of methods known in the art.

Uses Textile Applications

In another embodiment, the present invention relates to use of theendoglucanase of the invention in textile finishing processes, such asbio-polishing. Bio-polishing is a specific treatment of the yarn surfacewhich improves fabric quality with respect to handle and appearancewithout loss of fabric wettability. The most important effects ofbio-polishing can be characterized by less fuzz and pilling, increasedgloss/luster, improved fabric handle, increased durable softness andaltered water absorbency. Bio-polishing usually takes place in the wetprocessing during the manufacture of knitted and woven fabrics. Wetprocessing comprises such steps as, e.g., desizing, scouring, bleaching,washing, dying/printing and finishing. During each of these steps, thefabric is more or less subjected to mechanical action. In general, afterthe textiles have been knitted or woven, the fabric proceeds to anoptional desizing stage, followed by a scouring stage, etc. Desizing isthe act of removing size from textiles. Prior to weaving on mechanicallooms, warp yarns are often coated with size consisting of starch orstarch derivatives in order to increase their tensile strength. Afterweaving, the size coating must be removed before further processing ofthe fabric in order to ensure a homogeneous and wash-proof result. Inthe scouring process impurities are removed from the fabric. Theendoglucanase of the invention can advantageously be used in thescouring of cellulosic and cotton textiles, as well as bast fibers andmay improve efficiency of removal of impurities.

One of the most commonly used methods for delivering durable press tocellulosic textiles is via finishing with cellulose crosslinkingchemistry. Crosslinking immobilizes cellulose at a molecular level andsubstantially reduces shrinking and wrinkling of cellulosic garments.Treatment of durable press treated cellulosic textiles with theendo-glucanase of the invention may result in a selective relaxation ofstressed regions to minimize edge abrasion.

Additionally, the endoglucanase of the invention can be used toefficiently remove excess carboxymethyl cellulose-based print paste fromtextile and equipment used in the printing process.

It is known that in order to achieve the effects of bio-polishing, acombination of cellulolytic and mechanical action is required. It isalso known that “super-softness” is achievable when the treatment with acellulase is combined with a conventional treatment with softeningagents. It is contemplated that use of the endoglucanase of theinvention and of combinations of this enzyme with other enzymes forbio-polishing of cellulosics (natural and manufactured cellulosics,fabrics, garments, yarns, and fibers) is advantageous, e.g., a morethorough polishing can be achieved. It is believed that bio-polishingmay be obtained by applying the method described, e.g., in WO 93/20278.It is further contemplated that the endoglucanase of the invention canbe applied to simultaneous or sequential textile wet processes,including different combinations of desizing, scouring, bleaching,bio-polishing, dyeing, and finishing.

Stone-Washing

It is known that a “stone-washed” look (localized abrasion of the color)in dyed fabric, especially in denim fabric or jeans, can be providedeither by washing the denim or jeans made from such fabric in thepresence of pumice stones to provide the desired localized lightening ofthe color of the fabric or by treating the fabric enzymatically, inparticular with cellulytic enzymes. The treatment with an endoglucanaseof the present invention, alone or in combination with other enzymes,may be carried out either alone such as disclosed in U.S. Pat. No.4,832,864, together with a smaller amount of pumice than required in thetraditional process, or together with perlite such as disclosed in WO95/09225. Treatment of denim fabric with the endoglucanase of theinvention may reduce backstaining compared to conventional methods.

Biomass Degradation

The enzyme or the enzyme composition according to the invention may beapplied advantageously, e.g., as follows:

-   -   For debarking, i.e., pre-treatment with hydrolytic enzymes which        may partly degrade the pectin-rich cambium layer prior to        debarking in mechanical drums resulting in advantageous energy        savings.    -   For defibration (refining or beating), i.e., treatment of        material containing cellulosic fibers with hydrolytic enzymes        prior to the refining or beating which results in reduction of        the energy consumption due to the hydrolysing effect of the        enzymes on the surfaces of the fibers.    -   For fiber modification, i.e., improvement of fibre properties        where partial hydrolysis across the fibre wall is needed which        requires deeper penetrating enzymes (e.g., in order to make        coarse fibers more flexible).    -   For drainage: The drainability of papermaking pulps may be        improved by treatment of the pulp with hydrolysing enzymes. Use        of the enzyme or enzyme composition of to the invention may be        more effective, e.g., result in a higher degree of loosening        bundles of strongly hydrated micro-fibrils in the fines fraction        that limits the rate of drainage by blocking hollow spaces        between the fibers and in the wire mesh of the paper machine.

The treatment of lignocellulosic pulp may, e.g., be performed asdescribed in WO 93/08275, WO 91/02839 and WO 92/03608.

Laundry

The enzyme or enzyme composition of the invention may be useful in adetergent composition for household or industrial laundering of textilesand garments, and in a process for machine wash treatment of fabricscomprising treating the fabrics during one or more washing cycle of amachine washing process with a washing solution containing the enzyme orenzyme preparation of the invention.

Typically, the detergent composition used in the washing processcomprises conventional ingredients such as surfactants (anionic,nonionic, zwitterionic, amphoteric), builders, bleaches (perborates,percarbonates or hydrogen peroxide) and other ingredients, e.g., asdescribed in WO 97/01629 which is hereby incorporated by reference inits entirety.

Detergent Applications

The enzyme of the invention may be added to and thus become a componentof a detergent composition.

The detergent composition of the invention may for example be formulatedas a hand or machine laundry detergent composition including a laundryadditive composition suitable for pre-treatment of stained fabrics and arinse added fabric softener composition, or be formulated as a detergentcomposition for use in general household hard surface cleaningoperations, or be formulated for hand or machine dishwashing operations,especially for automatic dish washing (ADW).

The endo-beta-1,4-glucanase of the invention provides advantages such asimproved stain removal and decreased soil redeposition. Certain stains,for example certain food stains, contain beta-glucans which makecomplete removal of the stain difficult to achieve. Also, the cellulosicfibres of the fabrics may possess, particularly in the “non-crystalline”and surface regions, beta-glucan polymers that are degraded by thisenzyme. Hydrolysis of such beta-glucans, either in the stain or on thefabric, during the washing process decreases the binding of soils ontothe fabrics.

Household laundry processes are carried out under a range of conditions.Commonly, the washing time is from 5 to 60 minutes and the washingtemperature is in the range 15-60° C., most commonly from 20-40° C. Thewashing solution is normally neutral or alkaline, most commonly with pH7-10.5. Bleaches are commonly used, particularly for laundry of whitefabrics. These bleaches are commonly the peroxide bleaches, such assodium perborate, sodium percarbonate or hydrogen peroxide.

In a specific aspect, the invention provides a detergent additivecomprising the enzyme of the invention. The detergent additive as wellas the detergent composition may comprise one or more other enzymes suchas a protease, a lipase, a cutinase, an amylase, a carbohydrase, acellulase, a pectinase, a mannanase, an arabinase, a galactanase, axylanase, an oxidase, e.g., a laccase, and/or a peroxidase.

In general the properties of the chosen enzyme(s) should be compatiblewith the selected detergent, (i.e., pH-optimum, compatibility with otherenzymatic and non-enzymatic ingredients, etc.), and the enzyme(s) shouldbe present in effective amounts.

Proteases: Suitable proteases include those of animal, vegetable ormicrobial origin. Microbial origin is preferred. Chemically modified orprotein engineered mutants are included. The protease may be a serineprotease or a metalloprotease, preferably an alkaline microbial proteaseor a trypsin-like protease. Examples of alkaline proteases aresubtilisins, especially those derived from Bacillus, e.g., subtilisinNovo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 andsubtilisin 168 (described in WO 89/06279). Examples of trypsin-likeproteases are trypsin (e.g., of porcine or bovine origin) and theFusarium protease described in WO 89/06270 and WO 94/25583.

Examples of useful proteases are the variants described in WO 92/19729,WO 98/20115, WO 98/20116, and WO 98/34946, especially the variants withsubstitutions in one or more of the following positions: 27, 36, 57, 76,87, 97, 101, 104, 120, 123, 167, 170, 194, 206, 218, 222, 224, 235 and274.

Preferred commercially available protease enzymes include Relase®,Alcalase®, Savinase®, Primase®, Everlase®, Esperase®, Ovozyme®,Coronase®, Polarzyme® and Kannase® (Novozymes A/S), Maxatase™, Maxacal™,Maxapem™, Properase™, Purafect™, Purafect OxP™, FN2™, FN3™, FN4™ andPurafect Prime™ (Genencor International, Inc.), BLAP X and BLAP S(Henkel).

Lipases: Suitable lipases include those of bacterial or fungal origin.Chemically modified or protein engineered mutants are included. Examplesof useful lipases include lipases from Humicola (synonym Thermomyces),e.g., from H. lanuginosa (T. lanuginosus) as described in EP 258 068 andEP 305 216 or from H. insolens as described in WO 96/13580, aPseudomonas lipase, e.g., from P. alcaligenes or P. pseudoalcaligenes(EP 218 272), P. cepacia (EP 331 376), P. stutzeri (GB 1,372,034), P.fluorescens, Pseudomonas sp. strain SD 705 (WO 95/06720 and WO96/27002), P. wisconsinensis (WO 96/12012), a Bacillus lipase, e.g.,from B. subtilis (Dartois et al. (1993), Biochemica et Biophysica Acta,1131, 253-360), B. stearothermophilus (JP 64/744992) or B. pumilus (WO91/16422).

Other examples are lipase variants such as those described in WO92/05249, WO 94/01541, EP 407 225, EP 260 105, WO 95/35381, WO 96/00292,WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO 97/04079 and WO97/07202. Preferred commercially available lipase enzymes includeLipolase™ and Lipolase Ultra™ (Novozymes A/S).

Amylases: Suitable amylases (α and/or β) include those of bacterial orfungal origin. Chemically modified or protein engineered mutants areincluded. Amylases include, for example, α-amylases obtained fromBacillus, e.g., a special strain of B. licheniformis, described in moredetail in GB 1,296,839.

Examples of useful amylases are the variants described in WO 94/02597,WO 94/18314, WO 96/23873, and WO 97/43424, especially the variants withsubstitutions in one or more of the following positions: 15, 23, 105,106, 124, 128, 133, 154, 156, 181, 188, 190, 197, 202, 208, 209, 243,264, 304, 305, 391, 408, and 444.

Commercially used amylases are Duramyl®, Termamyl®, Stainzyme®,Fungamyl® and BAN® (Novozymes A/S), Rapidase™, Purastar™ and PurastarOxAm™ (from Genencor International Inc.).

Cellulases: Other suitable cellulases include those of bacterial orfungal origin. Chemically modified or protein engineered mutants areincluded. Suitable cellulases include cellulases from the generaBacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, e.g.,the fungal cellulases produced from Humicola insolens, Myceliophthorathermophila and Fusarium oxysporum disclosed in U.S. Pat. No. 4,435,307,U.S. Pat. No. 5,648,263, U.S. Pat. No. 5,691,178, U.S. Pat. No.5,776,757 and WO 89/09259.

Especially suitable cellulases are the alkaline or neutral cellulaseshaving color care benefits. Examples of such cellulases are cellulasesdescribed in EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, WO98/08940. Other examples are cellulase variants such as those describedin WO 94/07998, EP 0 531 315, U.S. Pat. No. 5,457,046, U.S. Pat. No.5,686,593, U.S. Pat. No. 5,763,254, WO 95/24471, WO 98/12307 and WO99/01544.

Commercially available cellulases include Celluzyme™, Renozyme® andCarezyme™ (Novozymes A/S), Clazinase™, and Puradax HA™ (GenencorInternational Inc.), and KAC-500(B)™ (Kao Corporation).

Peroxidases/Oxidases: Suitable peroxidases/oxidases include those ofplant, bacterial or fungal origin. Chemically modified or proteinengineered mutants are included. Examples of useful peroxidases includeperoxidases from Coprinus, e.g., from C. cinereus, and variants thereofas those described in WO 93/24618, WO 95/10602, and WO 98/15257.

Commercially available peroxidases include Guardzyme™ (Novozymes A/S).

Hemicellulases: Suitable hemicellulases include those of bacterial orfungal origin. Chemically modified or protein engineered mutants areincluded. Suitable hemicellulases include mannanase, lichenase,xylanase, arabinase, galactanase acetyl xylan esterase, glucorunidase,ferulic acid esterase, coumaric acid esterase and arabinofuranosidase asdescribed in WO 95/35362. Suitable mannanases are described in WO99/64619.

The detergent enzyme(s) may be included in a detergent composition byadding separate additives containing one or more enzymes, or by adding acombined additive comprising all of these enzymes. A detergent additiveof the invention, i.e., a separate additive or a combined additive, canbe formulated, e.g., as a granulate, a liquid, a slurry, etc. Preferreddetergent additive formulations are granulates, in particularnon-dusting granulates, liquids, in particular stabilized liquids, orslurries.

Non-dusting granulates may be produced, e.g., as disclosed in U.S. Pat.Nos. 4,106,991 and 4,661,452 and may optionally be coated by methodsknown in the art. Examples of waxy coating materials are poly(ethyleneoxide) products (polyethyleneglycol, PEG) with mean molar weights of1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethyleneoxide units; ethoxylated fatty alcohols in which the alcohol containsfrom 12 to 20 carbon atoms and in which there are 15 to 80 ethyleneoxide units; fatty alcohols; fatty acids; and mono- and di- andtriglycerides of fatty acids. Examples of film-forming coating materialssuitable for application by fluid bed techniques are given in GB1483591. Liquid enzyme preparations may, for instance, be stabilized byadding a polyol such as propylene glycol, a sugar or sugar alcohol,lactic acid or boric acid according to established methods. Protectedenzymes may be prepared according to the method disclosed in EP 238,216.

The detergent composition of the invention may be in any convenientform, e.g., a bar, a tablet, a powder, a granule, a paste or a liquid. Aliquid detergent may be aqueous, typically containing up to 70% waterand 0-30% organic solvent, or non-aqueous.

The detergent composition comprises one or more surfactants, which maybe non-ionic including semi-polar and/or anionic and/or cationic and/orzwitterionic. The surfactants are typically present at a level of from0.1% to 60% by weight.

When included therein the detergent will usually contain from about 1%to about 40% of an anionic surfactant such as linearalkylbenzenesulfonate, alpha-olefinsulfonate, alkyl sulfate (fattyalcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate,alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid orsoap.

When included therein the detergent will usually contain from about 0.2%to about 40% of a non-ionic surfactant such as alcohol ethoxylate,nonylphenol ethoxylate, alkylpolyglycoside, alkyldimethylamineoxide,ethoxylated fatty acid monoethanolamide, fatty acid monoethanolamide,polyhydroxy alkyl fatty acid amide, or N-acyl N-alkyl derivatives ofglucosamine (“glucamides”).

The detergent may contain 0-65% of a detergent builder or complexingagent such as zeolite, diphosphate, triphosphate, phosphonate,carbonate, citrate, nitrilotriacetic acid, ethylenediaminetetraaceticacid, diethylenetriaminepentaacetic acid, alkyl- or alkenylsuccinicacid, soluble silicates or layered silicates (e.g., SKS-6 from Hoechst).

The detergent may comprise one or more polymers. Examples arecarboxymethyl-cellulose, poly(vinylpyrrolidone), poly(ethylene glycol),poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole),polycarboxylates such as polyacrylates, maleic/acrylic acid copolymersand lauryl methacrylate/acrylic acid copolymers.

The detergent may contain a bleaching system which may comprise a H₂O₂source such as perborate or percarbonate which may be combined with aperacid-forming bleach activator such as tetraacetylethylenediamine ornonanoyloxybenzenesulfonate. Alternatively, the bleaching system maycomprise peroxyacids of, e.g., the amide, imide, or sulfone type.

The enzyme(s) of the detergent composition of the invention may bestabilized using conventional stabilizing agents, e.g., a polyol such aspropylene glycol or glycerol, a sugar or sugar alcohol, lactic acid,boric acid, or a boric acid derivative, e.g., an aromatic borate ester,or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid,and the composition may be formulated as described in, e.g., WO 92/19709and WO 92/19708.

The detergent may also contain other conventional detergent ingredientssuch as, e.g., fabric conditioners including clays, foam boosters, sudssuppressors, anti-corrosion agents, soil-suspending agents, anti-soilredeposition agents, dyes, bacteriocides, optical brighteners,hydrotropes, tarnish inhibitors, or perfumes.

In the detergent compositions any enzyme, in particular the enzyme ofthe invention, may be added in an amount corresponding to 0.01-100 mg ofenzyme protein per litre of wash liquor, preferably 0.05-5 mg of enzymeprotein per litre of wash liquor, in particular 0.1-1 mg of enzymeprotein per litre of wash liquor.

The enzyme of the invention may additionally be incorporated in thedetergent formulations disclosed in WO 97/07202 which is herebyincorporated as reference.

Signal Peptide and Propeptide

The present invention also relates to nucleic acid constructs comprisinga gene encoding a protein operably linked to a nucleotide sequenceencoding a signal peptide, wherein the gene is foreign to the nucleotidesequence encoding a signal peptide.

The present invention also relates to recombinant expression vectors andrecombinant host cells comprising such nucleic acid constructs.

The present invention also relates to methods for producing a proteincomprising (a) cultivating such a recombinant host cell under conditionssuitable for production of the protein; and (b) recovering the protein.

The first and second nucleotide sequences may be operably linked toforeign genes individually with other control sequences or incombination with other control sequences. Such other control sequencesare described supra. As described earlier, where both signal peptide andpropeptide regions are present at the amino terminus of a protein, thepropeptide region is positioned next to the amino terminus of a proteinand the signal peptide region is positioned next to the amino terminusof the propeptide region.

The protein may be native or heterologous to a host cell. The term“protein” is not meant herein to refer to a specific length of theencoded product and, therefore, encompasses peptides, oligopeptides, andproteins. The term “protein” also encompasses two or more polypeptidescombined to form the encoded product. The proteins also include hybridpolypeptides which comprise a combination of partial or completepolypeptide sequences obtained from at least two different proteinswherein one or more may be heterologous or native to the host cell.Proteins further include naturally occurring allelic and engineeredvariations of the above mentioned proteins and hybrid proteins.

Preferably, the protein is a hormone or variant thereof, enzyme,receptor or portion thereof, antibody or portion thereof, or reporter.In a more preferred aspect, the protein is an oxidoreductase,transferase, hydrolase, lyase, isomerase, or ligase. In an even morepreferred aspect, the protein is an aminopeptidase, amylase,carbohydrase, carboxypeptidase, catalase, cellulase, chitinase,cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase,alpha-galactosidase, beta-galactosidase, glucoamylase,alpha-glucosidase, beta-glucosidase, invertase, laccase, lipase,mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase,phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease,transglutaminase or xylanase.

The gene may be obtained from any prokaryotic, eukaryotic, or othersource.

The present invention is further described by the following exampleswhich should not be construed as limiting the scope of the invention.

EXAMPLES

Chemicals used as buffers and substrates were commercial products of atleast reagent grade.

Endoglucanase Activity Assay Materials:

Berol 537, nonionic surfactant supplied by Akzo Nobel, or similar.Cellazyme C tablets, supplied by Megazyme International, Ireland.Glass microfiber filters, GF/C, 9 cm diameter, supplied by Whatman.

pH 9.5 Buffer Solution:

Dissolve 21.0 g of NaHCO₃ and 14.6 g of NaCl in about 900 ml ofdeionised water. Add 10 ml Berol 537 (nonionic surfactant supplied byAkzo Nobel). Adjust the pH to 9.5 by addition of 4 N NaOH. Then adjustthe final volume to 1000 ml.

Method:

In test tubes, mix 1 ml pH 9.5 buffer and 5 ml deionised water.

Add 100 microliters of the enzyme sample (or of dilutions of the enzymesample with known weight:weight dilution factor). Add 1 Cellazyme Ctablet into each tube, cap the tubes and mix on a vortex mixer for 10seconds. Place the tubes in a thermostated water bath, temperature 40°C. After 15, 30 and 45 minutes, mix the contents of the tubes byinverting the tubes, and replace in the water bath. After 60 minutes,mix the contents of the tubes by inversion and then filter through aGF/C filter. Collect the filtrate in clean tubes.

Measure Absorbance (A_(enz)) at 590 nm, with a spectrophotometer. Ablank value, A_(water), is determined by adding 100 microliters waterinstead of 100 microliters enzyme dilution.

Calculate A _(delta) =A _(enz) −A _(water).

A_(delta) must be <0.5. If higher results are obtained, repeat with adifferent enzyme dilution factor. Determine DF0.1, where DF0.1 is thedilution factor needed to give A_(delta)=0.1.

Unit Definition:

1 Endo-Beta-Glucanase activity unit (1 EBG) is the amount of enzyme thatgives A_(delta)=0.10, under the assay conditions specified above. Thus,for example, if a given enzyme sample, after dilution by a dilutionfactor of 100, gives A_(delta)=0.10, then the enzyme sample has anactivity of 100 EBG/g.

Temperature and pH optima of the endoglucanase are determined by runningthe activity assay at a range of different temperatures when the pH isfixed and vice versa a range of different pH's when the temperature isfixed.

Example 1 Screening for Novel Endoglucanase

A number of Bacillus strains were screened for production of alkalineendoglucanase by growing the bacteria on TY agar added 0.1%AZCL-betaglucan (barley, Megazyme). Strain ACE160 produced blue haloeson this substrate, the bacterium was identified by determination of apart of the 16S rDNA, and insertion of the sequence in the phylogenetictree showed that ACE160 represent a new species with the Bacillus group.

Example 2 Production of Full Length Endoglucanase Genomic LibraryConstruction

Chromosomal DNA from ACE160 was prepared by using standard molecularbiology techniques (Ausuble et al. 1995 “Current protocols in molecularbiology” Publ: John Wiley and sons). The prepared DNA was partiallycleaved with Sau3A and separated on an agarose gel. Fragments of 3 to 8kilobases were eluted and precipitated and resuspended in a suitablebuffer.

A genomic library was made by using the Stratagene ZAP Express™predigested Vector kit and Stratagene ZAP Express™ predigested Gigapack®cloning kit (Bam HI predigested) (Stratagene Inc., USA) following theinstructions/recommendations from the vendor. The resulting lambdaZAPlibrary comprised 38000 pfu (plaque forming units) of which 10000 werecollected for mass excision. The resulting 70000 E. coli colonies werepooled. The E. coli clone pool was diluted by mixing 100 microliterspool with 100 ml LB medium and plated out 100 microliters per agarplateon LB supplemented with 0.1% AZCL.betaglucan (barley, Megazyme) and 50micrograms/ml kanamycin, and incubated for 2-3 days. Among 1600-1800colonies per plate on 50 agarplates three colonies with blue haloes wereobtained. From these three colonies plasmid DNA was recovered andsequenced with vector primers.

By subsequent primer walking the entire nucleotide sequence of theendo-1,4-betaglucanase open reading frame (ORF) was characterized. Thethree colonies contained the same ORF shown as SEQ ID NO:1.

Production of the Full Length Endoglucanase

To produce the endo-1,4-betaglucanase, the gene was amplified fromchromosomal DNA of the wild type strain Bacillus sp. ACE160. The enzymeswere expressed using the indigenous trans membrane signal peptide.

Primers

ACE160-Bglu-Mlu1-4: (SEQ ID NO: 3) GATTAACGCGTTCCTCGTGCTGAGCACAGAGGACE160-Bglu-Sac1: (SEQ ID NO: 4) TTATGGAGCTCAAATCAACTCTAGGAGGCTG

The endo-1,4-betaglucanase gene was amplified as a ca. 2500 nt PCRproduct. The primers ACE160-Bglu-Sac1 and ACE160-Bglu-Mlu1-4 were used.Template DNA was chromosomal DNA of Bacillus sp. ACE160. The PCR productwas recovered using Qiaquick™ spin columns as recommended (Qiagen,Germany). The quality of the isolated template was evaluated by agarosegel electrophoresis. PCR was run in the following protocol: 94° C., 2minutes 40 cycles of [94° C. for 30 seconds, 52° C. for 30 seconds, 68°C. for 1 minute] completed with 68° C. for 10 minutes. PCR product wasanalysed on a 1% agarose gel in TAE buffer stained with Ethidium bromideto confirm a single band of the correct size. The PCR product wasdigested with restriction enzymes Sad and Mlu1 and purified on GFX™ PCRand Gel Band Purification Kit (Amerham Biosciences).

The digested and purified PCR fragment was ligated to the Sac I and MluI digested plasmid pDG268NeoMCS-PramyQ/PrcryIII/cryIIIAstab/Sav (U.S.Pat. No. 5,955,310).

The ligation mixture was used for transformation into E. coli TOP10F′(Invitrogen BV, The Netherlands) and several colonies were selected forminiprep (QIAprep® spin, QIAGEN GmbH, Germany). The purified plasmidswere checked for insert before transformation into Bacillus subtilisstrain TH1 (TH1 is a Bacillus subtilis strain (amy-,spo-,apr-,npr-),that has been modified by insertion of a construct, from the strain DN3(Noone et al., 2000, J. Bacteriol. 182(6): 1592-1599) by transformationand selection for erytromycin. The changed genotype is: ykdA::pDN3(PykdA-lacZ Pspac-ykdA) Ermr. TH1 contains the following features: thefull ykdA promoter is fused to the LacZ reporter gene. In addition theykdA gene is placed under control of the IPTG-inducible Pspac promoter,so the ykdA gene no longer has it's naturally regulation. The strain canbe used as host for expression clones and libraries and transformantsexpressing and secreting protein can be selected on plates containingX-gal and IPTG. TH1 can be maintained on LB agar +6 micrograms/mLerythromycin).

Transformed cells were plated on LB-PG agar plates, supplemented with 1%skim milk, 100 micrograms/L X-gal, 1 mM IPTG, 6 micrograms/mlchloramphenicol and 12 micrograms/ml erythromycin. The plated cells wereincubated over night at 37° C. and colonies with blue color and withoutclearing zone were picked, the correct insert was confirmed by PCR andnucleotide sequencing.

Example 3 Purification of the Endoglucanase from Bacillus sp. ACE160

The endoglucanase was purified from 670 ml fermentation broth from whichthe cells were removed by a combination of centrifugation and filtrationof the broth. The volume was adjusted to 2 l with deionised water andthe pH titrated to 8.5. This material was loaded on a Q-sepharose columnequilibrated with 25 mM Tris buffer pH 8.5. The enzyme was eluted by theapplication of a NaCl gradient in the same buffer and the fractionscontaining the endoglucanase were pooled. A portion of this pool wasfractionated on a S-200 gel filtration column with 100 mM sodium acetatepH 6 as the liquid phase. The fractions containing the endoglucanasewere pooled and concentrated about three times on an Amiconultrafiltration unit. The concentrate was analyzed by SDS PAGE, where aprotein band of app. 80 kD was obtained.

Example 4 Wash Performance of Endoglucanase from Bacillus sp. ACE160

This procedure is used to determine the “enzyme detergency benefit”.

The wash tests are made by washing samples of soiled cotton fabric andsamples of clean cotton fabric, both together, in a small-scale washtest apparatus. After the washing the soil on the cotton fabric isevaluated by light reflectance. Both the originally soiled cotton fabricand the originally clean cotton fabric samples are evaluated.

Cotton fabric: #2003 white woven 100% cotton fabric, supplied byTanigashira, 4-11-15 Komatsu Yodogawa-ku, Osaka, 533-0004, Japan. Thenew cotton fabric is pre-washed three times before use in the wash test.The pre-washing is done using a European household front-loader washingmachine, and using a standard 40° C. wash process. LAS (Surfac® SDBS80sodium alkylbenzene sulfonate, 80%) is added to the wash water atconcentration 0.5 g per liter and the wash solution pH is adjusted to 10by addition of sodium carbonate. After the pre-washing the fabric isdried in a tumbler drier. Swatches of the pre-washed cotton fabric, size5 cm×5 cm, weight approximately 0.3 g each, are cut out and theseswatches are used for the wash tests.

Soiled cotton swatches: These are prepared from the 5 cm×5 cm swatchesdescribed above. Soiled swatches are made using beta-glucan (mediumviscosity, from barley, supplied by Megazymes International, Ireland)and carbon black (“carbon for detergency tests”, supplied by SentakuKagaku Kyokai, Tokyo, Japan). Dissolve about 0.67 g of beta-glucan in100 ml tap water by stirring and warming to >50° C. Add 0.33 g carbonblack. Blend with an UltraTurrax T25 blender, speed 4000 rpm for 2minutes. Apply 250 microliters of the beta-glucan/carbon onto the centerof each swatch. Allow to dry overnight at room temperature.Wash tests: Three soiled swatches and three clean swatches are washed ina Mini-Terg-O-Tometer machine. The Mini-Terg-O-Tometer is a small-scaleversion of the Terg-O-Tometer test washing machine described in Jay C.Harris, “Detergency Evaluation and Testing”, Interscience PublishersLtd. (1954) pp. 60-61. The following conditions are used:

Beaker size 250 ml Wash solution volume 100 ml Wash temperature 40° C.Wash time 30 minutes Agitation 150 rpm

The detergent solutions are pre-warmed to 40° C. before starting thetest. The fabric and the enzymes are added at the start of the 30 minutewash period. After the wash, the fabric swatches are rinsed for 5minutes under running tap water, then spread out flat and allowed to airdry at room temperature overnight.

Instrumental evaluations: Light reflectance evaluation of the fabricswatches is done using a Macbeth Color Eye 7000 reflectancespectrophotometer. The measurements are made at 500 nm. The UV filter isnot included. Measurements are made on the front and back of eachswatch. The soiled swatches are measured in the centre of the soiledarea. Average results for reflectance (R, 500 nm) for the soiledswatches and for the clean swatches are then calculated from the sixmeasurements on each type.Detergent solutions: Detergent solutions are prepared as follows: Toprepare 1 liter of solution, dissolve in deionized water 0.5 g sodiumcarbonate and 1.0 g sodium hydrogen carbonate and add 2 ml of a solutioncontaining 117.8 g/l CaCl₂.2H₂0 and 54.3 g/l MgCl₂.6H₂0. Thiscalcium/magnesium addition provides a water hardness of 12° dH. Add 0.2g nonionic surfactant (Berol® 537, Akzo Nobel) and 0.5 g LAS (Surfac®SDBS80 sodium alkylbenzene sulfonate, 80%) and adjust the final volumeto 1 liter. Adjust the pH to pH 9.5±0.1 (by addition of sodium carbonateor 10% citric acid solution).Enzyme addition: The enzymes to be tested are pre-dissolved at knownconcentrations in water, and the required amount of enzyme is added tothe detergent solution at the start of the wash process.Calculation of enzyme detergency benefit: The enzyme detergency benefitis a measure of how much more clean the swatches, both the originallysoiled and the originally clean, become as a result of including enzymesin the wash test. The enzyme detergency benefit is calculated asfollows:

After the wash test the average R, 500 nm value for the soiled swatchesis R, soiled.

After the wash test the average R, 500 nm value for the clean swatchesis R, clean.

The enzyme detergency benefit from a wash test with enzymes is the sumof R, soiled +R, clean with enzymes minus the sum of R, soiled +R, cleanwith no added enzyme.

The enzyme detergency benefit value determined in this way is a combinedmeasure both of the removal of soil from the fabric and of theredeposition of soil onto the fabric. Thus the enzyme detergency benefitvalue can have values that are negative or positive. The enzymedetergency benefit value can be used to compare the performance ofdifferent enzymes. The highest positive detergency benefit value is thepreferred result. For comparison, the wash performance of theendoglucanase from Bacillus sp. ACE160 was compared with of the washperformance of the prior art Bacillus endoglucanase MB1181-7 disclosedin WO 2002/099091.

Results:

Enzyme activity in wash solution Enzyme Detergency Benefit ACE160, 6 EBGper liter 28.1 ACE160, 12 EBG per liter 29.9 MB1181-7, 6 EBG per liter15.2 MB1181-7, 12 EBG per liter 22.2

The results show that the endoglucanase from Bacillus sp. ACE160 gives ahigher Enzyme Detergency Benefit than the known endoglucanase.

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

1-20. (canceled)
 21. An isolated polypeptide having endoglucanaseactivity, selected from the group consisting of: a) a polypeptide withat least 95% sequence identity to the sequence of amino acids 1 to 759of SEQ ID NO: 2; b) a polypeptide which is encoded by a polynucleotidewhich hybridizes under high stringency conditions with (i) nucleotides100 to 2376 of SEQ ID NO: 1, (ii) nucleotides 193 to 1041 of SEQ ID NO:1, or (iii) the full length complementary strand of (i) or (ii), whereinthe high stringency conditions are defined as prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 50% formamide, following standardSouthern blotting procedures, following by washing three times each for15 minutes using 2×SSC, 0.2% SDS at 65° C.; and c) a fragment of aminoacids 1 to 759 of SEQ ID NO: 2 having endoglucanase activity.
 22. Thepolypeptide of claim 21, which has at least 95% sequence identity to thesequence of amino acids 1 to 759 of SEQ ID NO:
 2. 23. The polypeptide ofclaim 21, which has at least 97% sequence identity to the sequence ofamino acids 1 to 759 of SEQ ID NO:
 2. 24. The polypeptide of claim 21,which has 100% sequence identity to the sequence of amino acids 1 to 759of SEQ ID NO:
 2. 25. The polypeptide of claim 21, which comprises thesequence of amino acids 1 to 759 of SEQ ID NO:
 2. 26. The polypeptide ofclaim 21, which is encoded by a polynucleotide which hybridizes underthe high stringency conditions with (i) nucleotides 100 to 2376 of SEQID NO: 1, (ii) nucleotides 193 to 1041 of SEQ ID NO: 1, or (iii) thefull length complementary strand of (i) or (ii).
 27. The polypeptide ofclaim 21, which is encoded by a polynucleotide which hybridizes undervery high stringency conditions with (i) nucleotides 100 to 2376 of SEQID NO: 1, (ii) nucleotides 193 to 1041 of SEQ ID NO: 1, or (iii) thefull length complementary strand of (i) or (ii), wherein the very highstringency conditions are defined as prehybridization and hybridizationat 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denaturedsalmon sperm DNA, and 50% formamide, following standard Southernblotting procedures, following by washing three times each for 15minutes using 2×SSC, 0.2% SDS at 70° C.
 28. The polypeptide of claim 21,which is a fragment of amino acids 1 to 759 of SEQ ID NO: 2 havingendoglucanase activity.
 29. The polypeptide of claim 21, which has atleast one of the following properties: a) a pl of 4.4, b) a pH optimumof 9, c) a temperature optimum of 40° C., or d) stability at pH from 5to 10.5.
 30. A composition comprising a polypeptide of claim
 21. 31. Thecomposition of claim 30, which further comprises one or more enzymesselected from the group consisting of alpha-amylases, cellulases,cutinases, glucoamylases, hemicellulases, laccases, ligninases, lipases,mannanases, oxidases, pectate lyases, pectin acetyl esterases,pectinases, pectin lyases, pectin methylesterases, peroxidases,phenoloxidases, polygalacturonases, proteases, pullulanases, reductases,rhamnogalacturonases, transglutaminases, xylanases, and xyloglucanases.32. A detergent composition comprising a polypeptide of claim 21 and asurfactant.
 33. A method for degrading cellulose-containing biomass,comprising treating the biomass with an effective amount of apolypeptide of claim
 21. 34. An isolated polypeptide havingendoglucanase activity, which comprises a catalytic domain with at least95% sequence identity to the sequence of amino acids 65 to 347 of SEQ IDNO: 2, wherein the catalytic domain has endoglucanase activity.
 35. Thepolypeptide of claim 34, wherein the catalytic domain has at least 98%sequence identity to the sequence of amino acids 65 to 347 of SEQ ID NO:2.
 36. The polypeptide of claim 34, wherein the catalytic domain has100% sequence identity to the sequence of amino acids 65 to 347 of SEQID NO:
 2. 37. The polypeptide of claim 34, wherein the catalytic domaincomprises the sequence of amino acids 65 to 347 of SEQ ID NO:
 2. 38. Acomposition comprising a polypeptide of claim
 34. 39. The composition ofclaim 38, which further comprises one or more enzymes selected from thegroup consisting of alpha-amylases, cellulases, cutinases,glucoamylases, hemicellulases, laccases, ligninases, lipases,mannanases, oxidases, pectate lyases, pectin acetyl esterases,pectinases, pectin lyases, pectin methylesterases, peroxidases,phenoloxidases, polygalacturonases, proteases, pullulanases, reductases,rhamnogalacturonases, transglutaminases, xylanases, and xyloglucanases.40. A detergent composition comprising a polypeptide of claim 34 and asurfactant.
 41. A method for degrading cellulose-containing biomass,comprising treating the biomass with an effective amount of apolypeptide of claim
 34. 42. An isolated polypeptide havingendoglucanase activity, which comprises a catalytic domain and acarbohydrate binding module, wherein the carbohydrate binding module hasat least 95% sequence identity to the sequence of amino acids 368 to 569of SEQ ID NO: 2 and has carbohydrate binding activity.
 43. Thepolypeptide of claim 42, wherein the carbohydrate binding module has atleast 97% sequence identity to the sequence of amino acids 368 to 569 ofSEQ ID NO:
 2. 44. The polypeptide of claim 42, wherein the carbohydratebinding module has 100% sequence identity to the sequence of amino acids368 to 569 of SEQ ID NO:
 2. 45. The polypeptide of claim 42, wherein thecarbohydrate binding module comprises the sequence of amino acids 368 to569 of SEQ ID NO:
 2. 46. A composition comprising a polypeptide of claim42.
 47. The composition of claim 46, which further comprises one or moreenzymes selected from the group consisting of alpha-amylases,cellulases, cutinases, glucoamylases, hemicellulases, laccases,ligninases, lipases, mannanases, oxidases, pectate lyases, pectin acetylesterases, pectinases, pectin lyases, pectin methylesterases,peroxidases, phenoloxidases, polygalacturonases, proteases,pullulanases, reductases, rhamnogalacturonases, transglutaminases,xylanases, and xyloglucanases.
 48. A detergent composition comprising apolypeptide of claim 42 and a surfactant.
 49. A method for degradingcellulose-containing biomass, comprising treating the biomass with aneffective amount of a polypeptide of claim 42.